The archetypal gene transfer agent RcGTA is regulated via direct interaction with the enigmatic RNA polymerase omega subunit

Gene transfer agents (GTAs) are small virus-like particles that indiscriminately package and transfer any DNA present in their host cell, with clear implications for bacterial evolution. The first transcriptional regulator that directly controls GTA expression, GafA, was recently discovered, but its mechanism of action has remained elusive. Here, we demonstrate that GafA controls GTA gene expression via direct interaction with the RNA polymerase omega subunit (Rpo-ω) and also positively autoregulates its own expression by an Rpo-ω-independent mechanism. We show that GafA is a modular protein with distinct DNA and protein binding domains. The functional domains we observe in Rhodobacter GafA also correspond to two-gene operons in Hyphomicrobiales pathogens. These data allow us to produce the most complete regulatory model for a GTA and point toward an atypical mechanism for RNA polymerase recruitment and specific transcriptional activation in the Alphaproteobacteria. [Display omitted] •Rhodobacter capsulatus transcriptional factor GafA is a multi-domain protein•Distal DNA-binding domains facilitate autoregulation and expression of RcGTA genes•GafA controls RNAP promoter selection via interaction with the omega subunit•Comparable gafA genes are present in the Hyphomicrobiales but split into two genes Sherlock and Fogg show that the Rhodobacter capsulatus transcriptional factor GafA is a multi-domain protein. The C terminus binds to its own promoter for autoregulation, the central region directly interacts with RNA polymerase via the omega subunit, and the whole protein is required for activation of the virus-like gene transfer agent.